Isolation and characterization of a Golgi-rich fraction from the adrenal medulla

153

Biochimica et Biophysica Acta, 421 (1976) 153--167 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27800 ISOLATION AND CHARACTERIZATION OF A GOLGI-RICH FRACTION FROM THE ADRENAL MEDULLA

J.M. TRIFAR(~ and A.C. DUERR Department of Pharmacology and Therapeutics, McGill University, Montreal (Canada)

(Received July llth, 1975)

Summary 1. A Golgi-rich fraction from bovine adrenal medulla was isolated by centrifugation through discontinuous sucrose density gradients. 2. The specific activity of UDPgalactose-N-acetylglucosamine galactosyl transferase was increased in this fraction. Therefore, this enzyme is a useful marker for Golgi in bovine adrenal medulla. 3. Golgi membranes were reasonably free from mitochondria, lysosomes, endoplasmic reticulum and chromaffin granules as shown by the relatively low activities of marker enzymes. 4. The negative staining techniques of electron microscopy revealed the presence of a system of tubules, vesicles and plate-like center regions which are similar to those structures previously described of the Golgi fraction isolated from the liver. 5. The specific activity of 5'-nucleotidase in the Golgi-rich fraction was 3.5 times greater than that in adrenal homogenates. Ho.wever, the subcellular distribution patterns of galactosyl transferase and 5'-nucleotidase were similar. The possibility that 5'-nucleotidase might be a conspicious c o m p o n e n t of the Golgi apparatus is discussed.

Introduction The adrenal medulla stores its secretory products in specific storage particles, the chromaffin granules. During stimulation, the granule membrane fuses with the plasmalema of the chromaffin cell and releases the soluble granule components (catecholamines, ATP, chromogranins and dopamine fi-hydroxylase) to the cell exterior by a process of exocytosis [1--3]. The granule membranes are then retained within the cell as empty vesicles [4--7]. Although there is much electron microscopic [8--10] and biochemical evidence [4-6,11] to support release by exocytosis, little is known about origin of the

154

granules and the ultimate fate of their membranes. The empty granules might be incorporated and digested by lysosomes [4] ; alternatively, they might move to the Golgi complex, fuse with it, and then be recharged with soluble granule components [ 12]. Electron microscopic observations have suggested the Golgi apparatus as the place of origin of chromaffin granules [ 8 , 1 3 ] , but there is no biochemical evidence that would substantiate this hypothesis. The origin of the storage granules must be established in order to understand the events involved in the secretory process. One approach to this problem might be the isolation and characterization of the Golgi complex of the adrenal medulla, followed by a comparative biochemical study between the membranes of the Golgi apparatus and those of the chromaffin granules. This paper describes a method for the isolation and characterization of a Golgi-rich fraction of the adrenal medulla. A preliminary account of some of these findings has already been given [ 14]. Materials and Methods

Subcellular fractionation of the adrenal medulla Bovine adrenal glands obtained from a slaughterhouse were kept on ice and the medulla were separated from the cortices. Each medulla was homogenized in 4 volumes of ice-cold 0.3 M sucrose (pH 7.0) in a motor-driven ADRENAL MEDULLA

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I Fig. 1. F l o w

diagram of the procedure followed for the isolation of the Golgi-rich fraction from the a d r e n a l m e d u l l a . F r a c t i o n 3 o f g r a d i e n t 1 is r e f e r ~ e d t o in t h e t e x t as " c r u d e G o l g i f r a c t i o n " . F u r t h e z d e t a i l s are d e s c r i b e d u n d e r M e t h o d s .

155 homogenizer (2750 rev./min) having a glass tube and teflon pestle (clearance 0.5 mm). The pestle was passed up and down three times. A low speed sediment was removed by centrifugation at 800 × g for 10 min and the supernatant fraction was recentrifuged one more time at the same speed and for the same period of time. The supernatant thus obtained was centrifuged at 20 000 × g for 20 min and the resulting sediment (crude granule fraction) was suspended in 4 ml of 0.3 M sucrose and layered on a gradient (gradient 1, Fig. 1) of the following composition (from top to bottom): 5, 6, 6, 6 and 10 ml of 0.8, 1.0, 1.2, 1.4 and 1.6 M sucrose (pH 7.0), respectively. The tube was then centrifuged in a SW 27 rotor (Beckman) at 113 000 × g for 70 min. Six fractions (number 1, 3 ml; numbers 2, 3, 4 and 5, 6 ml each and number 6, 10 ml) and a pellet (fraction number 7) were separated f r o m top to b o t t o m (Fig. 1). The pellet which contained chromaffin granules was resuspended in 4 ml of 1.6 M sucrose. The different fractions were assayed for protein and for catecholamines. The activities of enzymes which are characteristic markers of the different subcellular organelles were also measured in these fractions. The best resolution and separation were obtained when a "crude granule fraction" (20 000 × g sediment) prepared from 2--2.5 g of medulla was used. Fig. 1 shows all the steps followed in the preparation of the different subcellular fractions.

Preparation of the Golgi fractions The subcellular material which sedimented at the interfase between 0.8 and 0.1 M sucrose (fraction 3 of gradient 1, see Fig. 1) was collected from two similar gradients. These two fractions (crude Golgi fraction) were combined and the molarity of the preparation was adjusted to 1.1 M by adding sucrose; this was measured with the aid of a refractometer (Fisher Scientific Co.). The suspension was brought to a volume of 10 ml with 1.1 M sucrose and then layered on top of a second discontinuous sucrose gradient (gradient 2, Fig. 1) of the following composition (from top to bottom): 6, 6 and 8 ml of 1.25, 1.3 and 1.4 M sucrose (pH 7.0). 6 ml of 0.5 M sucrose were layered on top of the preparation. The tube (gradient 2, Fig. 1) was then centrifuged in a SW 27 rotor (Beckman) at 100 000 × g for 90 min. Five fr~ctions (top (T), 8.5 ml; Golgi (G), 2.5 ml; intermediate (I), 4 ml; endoplasmic reticulum (ER), 3 ml; and b o t t o m (B), 18 ml) were separated (Fig. 1) and assayed for protein and the different marker enzymes.

Analy tical procedures Protein was precipitated from the fractions by adding trichloroacetic acid to a final concentration of 10%. Protein was determined by the m e t h o d of Lowry et al. [15], using bovine serum albumin as standard. Catecholamines were assayed by the trihydroxyindole fluorometric method of Anton and Sayre [16]. Inorganic phosphate was determined according to the m e t h o d of Ames [17]; assays were carried out at two protein concentrations and the results averaged. Monoamine oxidase activity (MAO, EC 1.4.3.4) was measured by the m e t h o d of Wurtman and Axelrod [18]; [1-14C] tyramine hydrochloride (specific activity, 43.7 Ci/mol) was used as substrate and other modifications were

156 as previously described [ 19]. fi-Glucuronidase (EC 3.2.1.31) activity was determined by the method described by Gianetto and De Duve [20]. Dopamine fi-hydroxylase (EC 1.14.4.1) was measured by the method described by Viveros et al. [ 2 1 ] , using [ 3H] tyramine hydrochloride (specific activity 10 Ci/mmol) as substrate. Succinic dehydrogenase (EC 1.3.99.1) activity was determined by the method of Kuff and Schneider [22]. Glucose-6-phosphatase (EC 3.1.3.9) and 5'-nucleotidase (AMPase, EC 3.1.3.5) activities were measured by the methods of De Duve et al. [23] and Mitchell and Hawthorne [24], respectively. R N A was determined by the method described by Schneider [25]. UDPgalactose N-acetylglucosamine (galactosyl transferase, EC 2.4.1.67) was determined by a modification of the method of Fleisher et al. [26]. Samples (30--50 pg protein) of the different subcellular fractions were incubated for 60 min at 37°C in a total volume of 80 pl. The assay mixture (80 pl) contained the following substances (pmol): sodium cacodylate (pH 6.75), 6; 2-mercaptoethanol, 3; MnC12, 3; N-acetylglucosamine, 3; and uridine diphospho[~4C] galactose (UDP[ ~*C] Gal) uniformly labeled in the sugar moiety (specific activity 2 • ].06 cpm/pmol), 0.05. Triton X-100 was present in the incubation medium at the concentration of 0.6%. The addition of 6 pmol of EDTA in 20 pl of distilled water (pH 7.0) terminated the incubations. The tubes were cooled in an ice-water bath for a few minutes and the mixture was passed through a column (0.5 cm × 2 cm) of Dowex 2X-8 (200--400 mesh), in the C1- form. The column was then washed twice with 0.5 ml of distilled water (pH 7.0). All the effluents of the column were collected into scintillation vials containing 10 ml of "Aquasol" (New England Nuclear). The radioactivity was measured in a liquid scintillation spectrometer (Intertechnique) and the results were expressed as nmol of galactose transferred per mg protein per h. Control tubes containing all the above substances, except N-acetylglucosamine, were also run.

Electron microscopy The negative staining procedure was performed on a drop of the sample placed on a carbon-coated collodion filmed copper grid. A drop of 2% phosphotungstic acid brought to pH 7.2 by addition of KOH was then applied to the grid. The excess of stain was immediately removed by touching the edge of the grid with a filter paper. Electron microscopic examination of the samples was made using a Phillips EM 300 electron microscope.

Chemicals The chemicals were obtained from the following sources: N-acetylglucosamine, 2-mercaptoethanol, Triton X-100, sodium cacodylate, glucose 6-phosphate, adenosine monophosphate (AMP) and phenolphtaleinglucuronic acid (sodium salt) from Sigma Chemical Company; [ 3 H ] t y r a m i n e hydrochloride, [ 14 C] tyramine hydrochloride and uridine diphospho [ 14 C] galactose from New England Nuclear; acrylamide and N'N'-methylenebisacrylamide from Eastman Kodak Company; and disodium ethylene diamine tetra-acetate (EDTA) from Fisher Scientific Company.

157 Results

Isolation of a Golgi-rich fraction It is k n o w n from previous work that centrifugation of the low speed supernatants obtained from adrenal medullary homogenates at 20 000 × g for 20 min yielded a sediment (crude or large granule fraction) which, in addition to chromaffin granules, contains mitochondria, lysosomes and some, but n o t all, of the microsomal particles [27]. It is also known that chromaffin granules sediment below a layer of 1.6 M sucrose [28] and that empty chromaffin granules or their membranes, sediment at the top of the density gradients [5,29] and that they equilibrate at the level of 0.8 M sucrose [29]. Therefore, a discontinuous density gradient was prepared taking into consideration all this information. This gradient, which is described as gradient 1 under Methods, provided a good separation of the different subcellular components of the adrenal medulla (Figs. 1, 2 and 3). As expected the bulk of chromaftln granules was recovered in the sediment (fraction 7) as indicated by the distribution of catecholamines (Fig. 3) and of dopamine-fi-hydroxylase (Fig. 2). The specific activity of this enzyme was greater in fraction 2 than in fraction 7 (Fig. 2). This is due to the fact that fraction 2 contained empty chromaffin granules or their membranes and that the ratio of dopamine fi-hydroxylase to protein is greater in granule membranes than in intact chromaffin granules [30]. Mitochondria were mainly recovered in fractions 4 and 5 which corresponded to the interfaces between layers 1.0--1.2 and 1.2--1.4 M sucrose, respectively. This can be observed in Fig. 3 which shows the distribution of monoamine oxidase. A similar subcellular distribution was obtained when succinic dehydrogenase was used as a mitochondrial marker. Lysosomes evaluated by the distribution of ~-glucuronidase, sedimented at the level of fraction 5 and especially fraction 6 (Fig. 3).

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Fig. 3. A n a l y s e s o f t h e d i f f e r e n t s u b c e l l u l a r f r a c t i o n s o b t a i n e d f r o m t h e " c r u d e g r a n u l e f ~ a c t i o n " afte~ s u c c e s s i v e c e n t r i f u g a t i o n s on g r a d i e n t s 1 a n d 2. A " c r u d e g r a n u l e f r a c t i o n " ( 2 0 0 0 0 × g s e d i m e n t ) wa~ l a y e r e d o n s u c r o s e d e n s i t y g r a d i e n t 1 a n d c e n t r i f u g e d at 113 0 0 0 × t~ f o r 7 0 rain. F r a c t i o n 3 f r o m thi~, g r a d i e n t w a s c o l l e c t e d , its m o l a r i t y a d j u s t e d to 1.1 M a n d t h e n w a s l a y e r e d on g r a d i e n t 2. T h i s g r a d i e n t was c e n t r i f u g e d at 1 0 0 0 0 0 × g f o r 90 rain. F r a c t i o n s w e r e c o l l e c t e d f r o m g r a d i e n t s 1 a n d 2 a n d t h e y w e r e a s s a y e d f o r c a t e c h o l a m i n e s , ~ - g l u c u r o n i d a s e , m o n o a m i n e o x i d a s e a n d g a l a c t o s y l t r a n s f e r a s e as indic a t e d u n d e r M e t h o d s , p - H P H is t h e a b b r e v i a t i o n f o r p - h y d r o x y p h e n y l a c e t a l d e h y d e .

UDPgalactose N-acetylglucosamine galactosyl transferase has been used as a marker for Golgi membranes which have been isolated from liver [31,32], testis [32,33] and anterohypophysis [34] homogenates. Therefore, the different layers of gradient 1 were also examined for their content in galactosyl transferase. The fraction collected at the interface between the 0.8--1.0 M sucrose layers (fraction 3, Figs. 1, 2 and 3} had the greatest specific activity. The specific activity of the galactosyl transferase in this "crude Golgi fraction" was 6 times greater than that found in the adrenal homogenates (Table I). However, the activity of the enzyme in the "crude Golgi fraction" was 23% lower than that observed in the Golgi-rich fraction isolated from another secretory tissue, the adenohypophysis (Table I). It should also be noticed that the crude Golgi fraction sedimented immediately below the fraction containing the greatest specific activity of dopamine ~-hydroxylase. This can be observed in Fig. 2, which, in addition, shows that the distribution of 5'-nucleotidase and galactosyl transferase are similar. A further purification of the "crude Golgi fraction" (fraction 3, gradient 1) was made and the sucrose density gradient first described by Leelavathi et al. [31] for the isolation of the Golgi from rat liver was used for this purpose.

159

TABLE

I

UDPoGALACTOSE N-ACETYLGLUCOSAMINE IOUS SUBCELLULAR FRACTIONS

GALACTOSYL

TRANSFERASE

The figttres represent the mean + S.D. of the number of experiments G o l g i F r a c t i o n " r e f e r s t o f r a c t i o n 3 o f g r a d i e n t 1 ( s e e F i g . 1). Fraction

Homogenate

Crude Golgi

Galactosyl transferase (nmol galactose transferred/h/mg

indicated

ACTIVITY

in parenthesis.

IN VAR-

"Crude

protein)

Adrenal medulla

Adenohypophysis

9 . 4 -+ 0 . 6 (15)

1 0 . 2 +- 0 . 7 (17)

63.2 + 2.8

--

*

(17) Golgi

* From

145.1 + 7.1 (12)

82.5 + 6.5 (i0)

D . W . M c K e e l a n d L. J a r r e t [ 3 4 ] .

After centrifugation, five fractions were separated (see Fig. 1) and assayed for galactosyl transferase activity. The final Golgi preparation was recovered at the interface between 0.5 and 1.1 M sucrose (Fig. 1). This fraction showed, when compared to "crude Golgi fraction", a further increase (230%) in the specific activity of the galactosyl transferase (Fig. 3, Table I). In addition, the specific activity of the galactosyl transferase in this preparation was 76% greater than that reported for the preparation isolated from the adenohypophysis (Table I). The distribution of galactosyl transferase before and after centrifugation on gradient 1 is shown in Table II. The nuclear fraction (800 × g sediment) contained 28.6% of the total enzyme activity. This high percentage was due to the gentle homogenization procedure employed which left 20--25% of the cells unbroken. The percentage of these intact cells, which sedimented in the nuclear fraction, was calculated as described previously from the percentage of catecholamines contained in the low speed sediment [5]. The use of a much stronger homogenization procedure would decrease the number of unbroken cells in the nuclear fraction and, at the same time, it would destroy a large number of Golgi cisternae and tubules with the formation of microsome-like vesicles. This latter procedure would decrease much more the number of Golgi structures recovered at the level of layer 3 of the gradient 1 (crude Golgi). Therefore, for this reason we have sacrificed the yield in favor of morphology and purity of the preparation. Consequently, only 22.9% of the total galactosyl transferase activity was recovered in the crude Golgi fraction (Table II). The protein and galactosyl transferase distributions of the "crude Golgi fraction" after further resolution by means of the density gradient 2 are shown in Table III. The final Golgi fraction contained 24 and 60% of the protein and galactosyl transferase of the crude Golgi fraction. The total galactosyl transferase in the final Golgi fraction represents 14% of that found in the original homogenate. The c o n t e n t of RNA in the final Golgi preparation was 47.1 + 3.3 pg/mg protein (n = 6), and this represented 1.2 ± 0.1% (n = 6) of th'e total RNA content of the adrenal medulla. The degree of purity of this Golgi preparation

160 T A B L E II T O T A L G A L A C T O S Y L T R A N S F E R A S E A N D 5 ' - N U C L E O T I D A S E A C T I V I T I E S IN V A R I O U S SUBCELLULAR FRACTIONS OBTAINED FROM BOVINE ADRENAL MEDULLA T h e h o m o g e n a t e f i g u r e s c o r r e s p o n d to 2.5 g of fresh a d r e n a l m e d u l l a . G a l a c t o s y l t r a n s f e r a s e a n d 5'-nuc l e o t i d a s e a c t i v i t i e s are e x p r e s s e d as n m o l g a l a c t o s e t r a n s f e r r e d p e r h p e r f r a c t i o n a n d nrnol Pi r e l e a s e d p e r rain per fraction, respectively. Each figure represents the average of 4 separate e x p e r i m e n t s .

Fraction

Total protein (mg)

Homogenate Nuclear fraction * C r u d e g r a n u l e f r a c t i o n ** Post " C G F " s u p e r n a t a n t * * * F r a c t i o n 2, g r a d i e n t 1 Crude Golgi Mit o c h o n d r i a Chromaffin granules

134.5 31.4 47.6 36.3 5.2 4.2 8.1 8.9

Galactosyl transferase

5'-nucleoridase

Total activity

%

Total Activity

%

1264 361 417 250 50 290 72 5

100 28.6 33.0 19.8 4.0 22.9 6.0 0.4

1346 900 136 100 35 69 32 0.09

100 66.9 10.1 7.4 2.6 5.1 2.4 0.007

* S e d i m e n t o b t a i n e d a f t e r c e n t r i f u g a t i o n o f t h e h o m o g e n a t e a t 8 0 0 X g f o r 10 rain. T h i s c o n t a i n e d 2 0 - - 2 5 % of t h e cells u n b r o k e n ; this f i g u r e w a s c a l c u l a t e d f r o m t h e p e r c e n t a g e c h o l a m i n e c o n t a i n e d in t h i s f r a c t i o n . T h e c a t e c h o l a m i n e s s e d i m e n t e d at t h i s l o w s p e e d , t a i n e d w i t h i n u n b r o k e n cells [ 5 ] . ** S e d i m e n t o b t a i n e d a f t e r c e n t r i f u g a t i o n o f t h e p o s t n u c l e a r s u p r e n a t a n t at 20 0 0 0 × g f o r * * * S u p e r n a t a n t o b t a i n e d in t h e a b o v e c e n t r i f u g a t i o n .

fraction of cateare con20 m i n .

was also evaluated by measuring the specific activities of enzymes known to be markers for mitochondria, lysosomes, endoplasmic reticulum, plasma membrane, and chromaffin granules. These different enzymatic activities are shown in Table IV and, as expected from the results obtained with gradient 1 (Fig. 2), there was also here an increase in the specific activity of 5'-nucleotidase of the

T A B L E III SUBFRACTIONATION

OF THE CRUDE GOLGI FRACTION OF THE ADRENAL MEDULLA

F r a c t i o n 3 f r o m g r a d i e n t 1 ( c r u d e G o l g i f r a c t i o n ) w a s c o l l e c t e d , its m o l a r i t y a d j u s t e d to 1.1 M a n d t h e n w a s l a y e r e d o n g r a d i e n t 2. T h e g r a d i e n t w a s c e n t r i f u g e d a t 1 0 0 0 0 0 X g f o r 9 0 m i n . F r a c t i o n s w e r e obt a i n e d as o u t l i n e d in Fig. 1. G a l a c t o s y l t r a n s f e r a s e t o t a l a c t i v i t y is e x p r e s s e d as n m o l e s g a l a c t o s e t r a n s f e r r e d p e r h o u x p e r f r a c t i o n . E a c h f i g u r e r e p r e s e n t s t h e a v e r a g e of 3 s e p a r a t e e x p e r i m e n t s . Gaiactosyl transferase

Total protein mg

Recovery %

Total activity

Recovery %

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4.2

100

290

100

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162

Golgi fraction. The total activity of the 5'-nucleotidase in the h0mogenate and the distribution of this activity in the different subcellular fractions are shown in Table II. The highest percentage of the enzyme activity was re'covered in the 800 × g sediment, a fraction which contains nuclei, unbroken cells and plasma membranes. The specific activity of the 5'-nucleotidase in the nuclear fraction (28.7 nmol/mg protein/h) was higher than that found in the homogenate, and lower than that present in the Golgi fraction (Table IV). When the Golgi and endoplasmic reticulum fractions were compared, it was observed that, as expected, the endoplasmic reticulum was the richest in glucose-6-phosphatase activity and the poorest in galactosyl transferase activity (Table IV and Fig. 3). The ratio of galactosyl transferase to glucose-6-phosphatase activity in Golgi fraction was 46.3 + 1.1 (n = 6) times greater than that calculated for the endoplasmic reticulum. Furthermore, traces of dopamine fl-hydroxylase were detected in all Golgi preparations obtained by this procedure. The specific activity of the dopamine ~-hydroxylase in the Golgi preparation was 1/12 of that found in the chromaffin granule membranes (Table IV). A small galactosyl transferase activity was detected in 15 of the 18 chromaffin granule preparations tested (Table IV). Since this activity could occur if the granules were less than 0.5% contaminated with Golgi membranes, it seems the chromaffin granules have no significant endogenous galactosyl transferase activity.

Electron microscopy The electron microscopic examination of negative stained preparations of the Golgi fraction isolated by means of gradient 2 revealed the tubular nature of the cisternae (Fig. 4a and Fig. 5). There is an extensive system of peripheral tubules which seem to be connected with the plate-like center regions of the cisternae (Fig. 4a). The latter corresponds to the parallel Golgi cisternae seen in the intact cell [10]. The tubules seen in the preparations are of two types. Those close to the cisternae which are the smallest in diameter (260--320 A), are highly anas~omosed, especially in the periphery of the plate-like center regions (Figs. 4a and 5). The tubules situated far from the plate-like center regions are of a large diameter (350--500 £) and many of them ended in a vesicle-like structure (Figs. 4b and 5). These tubules are probably closely related to the maturing phase of the Golgi. Some areas of these Golgi preparations show collections of large tubules and isolated vesicles (Fig. 4b) which probably have separated from the cisternae during homogenization. This is quite possible since there are many plate-like center regions in which the peripheral fenestrae are missing (Fig. 4a). However, it is possible to see in the periphery of these plates the area from which the tubules have emerged (Fig. 4a). All preparations show a small number of mitochondria and this is in agreement with the low c o n t e n t in succinic dehydrogenase of these preparations {Table IV). Vesicles of dimensions between 0.1 and 0.2 pm are also seen in these Golgi preparations. It is difficult to determine the origin of these vesicles, except in the case of those vesicles which are attached to tubules (Fig. 5) or connected through tubules to the plate-like center regions of the cisternae (Fig. 4a). Finally, some cisternae may be entirely tubular as shown in Fig. 5, which also shows three or four stacked tubular cisternae (dyctyosome).

163

F i g . 4. E l e c t r o n m i c r o s c o p y o f G o l g i - r i c h f r a c t i o n s f r o m a d r e n a l m e d u l l a n e g a t i v e s t a i n e d w i t h 2 % P T A . C h a r a c t e r i s t i c f e a t u r e s o f t h e s e f r a c t i o n s a r e : a) (X 4 8 7 5 0 ) , t h e p r e s e n c e o f p l a t e - l i k e c e n t e r r e g i o n s ( P ) and an extensive system of peripheral tubules (Tu). The zones of connections between plates and tubules a r e i n d i c a t e d b y t h e s i n g l e a r r o w s . V e s i c l e s ( V e ) c a n b e also o b s e r v e d i n t h i s p r e p a r a t i o n , a n d o n e o f t h e s e v e s i c l e s is c o n n e c t e d t h r o u g h a t u b u l e t o t h e p l a t e - l i k e c e n t e r r e g i o n ( d o u b l e a r r o w ) , b ) ( X S 1 9 0 0 ) , a r e a s containing large tubules (Tub) and vesicles (Ve).

164

Fig. 5. E l e c t r o n m i c r o s c o p y o f a Golgiorich f r a c t i o n f r o m a d r e n a l m e d u l l a n e g a t i v e s t a i n e d w i t h 2% p h o s p h o t u n g s t i c acid. T h e p r e p a r a t i o n s h o w s large t u b u l e s ( T u b ) , vesicles ( V e ) , a n d a t u b u l a r c i s t e r n a e ( T u ) . T h r e e o r f o u r s t a c k e d t u b u l a r c i s t e r n a e a r e s h o w n (single a r r o w s ) . A vesicle c o n n e c t e d to the c i s t e r n a e t h r o u g h a t u b u l e is i n d i c a t e d b y t h e d o u b l e a r r o w .

Discussion The UDPgalactose-N-acetylglucosamine galactosyl transferase is one of the sugar transferases known to be present in the Golgi complex of different tissues. This e n z y m e has been previously used as a marker for Golgi membranes in subcellular fractionation studies carried out in liver, testes and hypophysis [ 3 1 - - 3 4 ] . We have f ound t ha t the adrenal medulla has a high c o n t e n t of galactosyl transferase and since we did n o t have any valid reason to suspect a different subcellular localization of the enzyme in this tissue, the galactosyl transferase was also used as a Golgi marker in our studies. The present study shows the isolation and characterization of a Golgi-rich fraction from the adrenal medulla. The specific activity of the galactosyl transferase in our final Golgi preparation was lower than that report ed for the Golgi fraction from rat liver [ 3 2 , 3 5 ] . However, the galactosyl transferase specific activity o f the adrenal preparation was 76% greater than that obtained from rat hypophysis [ 3 4 ] , endocrine tissue which shares with the adrenal medulla the c o m m o n feature of storing its hor m ones in subcellular granules.

165 The Golgi fraction isolated from the adrenal medulla has similar sedimentation properties as the fraction isolated from rat liver [31] and the morphology of the medullary Golgi fraction as evaluated by negative staining techniques seems also to be similar to that previously described for the liver Golgi fraction [31,35]. Both liver and medulla preparations consist of a system of tubules, vesicles and plate-like center regions. The origin of the vesicles present in our Golgi preparations is difficult to determine. They might have been part of the Golgi apparatus which became detached from the cisternae during the subcellular fractionation procedure, with only a few vesicles remaining connected through tubules to the plate-like Center regions. However, the presence of vesicles derived from the endoplasmic reticulum, plasma membrane or empty chromaffin granules cannot be ruled out. The low content in glucose-6-phosphatase of the Golgi preparation might indicate that a small amount of endoplasmic reticulum structures are present as contaminants, but, the possibility exists that this small amount of glucose-6phosphatase is indigenous to Golgi membranes, especially to those Golgi cisternae located in the forming phase of the Golgi apparatus [35]. The presence in the Golgi of dopamine ~-hydroxylase with a specific activity of 1/12 of that found in granule membranes might indicate a small degree of chromaffin granule membrane contamination, b u t here again, it is possible that dopamine ~-hydroxylase is a conspicuous c o m p o n e n t of the Golgi apparatus, especially if the chromaffin granules are derived from the Golgi complex. Furthermore, dopamine ~-hydroxylase is a glycoprotein [36,37] and the Golgi complex is the cell organelle where the glycosidation reactions necessary for glycoprotein synthesis take place [38]. Immunohistochemical studies performed on intact adrenal medullae would indicate whether or not dopamine ~-hydroxylase is a c o m p o n e n t of the Golgi membrane, and such studies are currently being carried out in our laboratory. 5'-Nucleotidase appears to be a c o n s t i t u e n t of Golgi membranes. This enzyme has been used as a marker for plasma membrane [39], and indeed, this enzyme has a very high specific activity (8.84 pmol/mg protein/h) in the plasma membrane fraction isolated from the adrenal medulla [40]. This plasma membrane fraction has also a very high content of Ca2÷-d.ependent ATPase (EC 3.6.1.3; 260.95 pmol/mg protein/h). The Golgi fraction isolated from the adrenal medulla has a Ca2÷-dependent ATPase value (n = 6) of 4.43 + 0.34 pmol/mg protein/h (unpublished observations) and a 5'-nucleotidase specific activity (2.1 pmol/mg protein/h) which, although higher than that found in the adrenal homogenate, is only 24% of that reported for plasma membrane when using the same technique of determination. It can be argued that the 5'-nucleotidase activity in the Golgi fraction represents a plasma membrane contamination of 24%. However, if this were the case, the Ca2÷-dependent ATPase activity in the Golgi fraction would be 24% (62.6 nmol/mg protein/h) of that found in plasma membrane. But, on the contrary, the specific activity of the Ca 2÷dependent ATPase in the Golgi fraction was only 1.7% of that reported for plasma membranes. Furthermore, our study showing the similarity between the subcellular distribution of galactosyl transferase and 5'-nucleotidase, would suggest that this latter enzyme is a true c o m p o n e n t of the Golgi apparatus rather than a contamination by plasma membrane. It has been recently demonstrated

166 by means of histochemical techniques carried out on intact cells that 5'-nucl~otidase is also present in the Golgi complex of the liver [41]. Moreover, the ratio of Ca2÷-dependent ATPase to 5'-nucleotidase, calculated from the published data [40], for the plasma membrane of the adrenal medulla is 29.5, whereas the same ratio for the Golgi membranes is only 2.1. All this evidence would indicate that 5'-nucleotidase is indeed a conspicuous component of the Golgi apparatus. The biochemical comparison of the Golgi membranes obtained by the method described here with those membranes prepared from chromaffin granules would be an important step in the understanding of the origin of chromaffin granules. Preliminary studies undertaken in our laboratory seem to indicate that there are marked biochemical differences between these two types of membranes. This would suggest that the chromaffin granules are n o t derived from the Golgi apparatus by an unspecific "membrane flow".

Ackno wledgements This work was supported by grants from the Medical Research Council of Cananda. A.C. Duerr held a RODA Scholarship. We wish to thank Dr. J. Lowenthal and Dr. B. Collier for their helpful comments. We are indebted to Dr. A.F. Graham ahd Mr. L. Guluzian of the Department of Biochemistry for the electron microscopy. We are also grateful to Miss C. Shorten and Mrs. C. Ulpian for their skilled technical assistance.

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